Synthesis and evaluation of small molecules bearing a benzyloxy substituent as novel and potent monoamine oxidase inhibitors

A new series of small molecules bearing a benzyloxy substituent have been designed, synthesized and evaluated for hMAO inhibitory activity in vitro.

Abstract

A new series of small molecules bearing a benzyloxy substituent have been designed, synthesized and evaluated for hMAO inhibitory activity in vitro. Most of the compounds were potent and selective MAO-B inhibitors, and were weak inhibitors of MAO-A. In particular, compounds 9e (IC₅₀ = 0.35 μM) and 10e (IC₅₀ = 0.19 μM) were the most potent MAO-B inhibitors, and exhibited the highest selectivity for MAO-B (9e, SI > 285.7-fold and 10e, SI = 146.8-fold). In addition, the structure–activity relationships for MAO-B inhibition indicated that electron-withdrawing groups in the open small molecules were more suitable for MAO-B inhibition, and substitutions at the benzyloxy of the open small molecules, particularly with the halogen substituted benzyloxy, were more favorable for MAO-B inhibition. Molecular docking studies have been done to explain the potent MAO-B inhibition of the open small molecules. Furthermore, the representative compounds 9e and 10e showed low neurotoxicity in SH-SY5Y cells in vitro. So the small molecules bearing the benzyloxy substituent could be used to develop promising drug candidates for the therapy of neurodegenerative diseases.

Introduction

Monoamine oxidase (EC 1.4.3.4; MAO), located in the peripheral tissues and the central nervous system (CNS), is a FAD-containing enzyme, which binds tightly to the outer mitochondrial membrane of glial, neuronal and other cells. MAOs could regulate and metabolize biogenic amines by oxidative deamination, such as serotonin, dopamine (DA) and epinephrine.1

On the account of differences in inhibitor specificity, immunological properties, substrate preference, amino acid sequences and tissue distribution, mammals contain two distinctive MAO enzymes, namely, MAO-A and MAO-B.2–6 The MAO-A preferentially metabolizes epinephrine, norepinephrine, and serotonin, and is selectively inhibited by clorgyline. Whereas the MAO-B is selectively inhibited by selegiline or rasagiline, and specifically deaminates β-phenethylamine.7,8 With an increasing number of studies into the X-ray crystal structures of the two MAOs, information about the pharmacophoric requirements and the selective interactions is useful to design potent and selective MAO inhibitors. The active sites of hMAOs are supposed to be the key structural differences because of the different shapes and volumes of the inhibitors/substrate binding pockets. The active site of hMAO-A is a single hydrophobic cavity, while hMAO-B has two distinct cavities: one is the so-called “substrate cavity”, which is connected to the flavin adenine dinucleotide cofactor (FAD) and the other smaller cavity called the “entrance cavity” is located towards the outside of the protein. These cavities of MAOs have narrow pockets, but in hMAO-B the Tyr 326 and Ile 199 residues act as a bottleneck and thus form a gate, which separates the region as two cavities.9,10

Since the MAOs could terminate the actions of neurotransmitter amines in the CNS, they are regarded as attractive targets to treat psychiatric and neurological disorders. Selective MAO-A inhibitors are effective in the therapy of depression,11 while selective MAO-B inhibitors have been applied alone or in combination to treat Alzheimer's and Parkinson's diseases.12 Thus specific MAO inhibitors are developed for the treatment of neurological disorders.

Hetero-benzofused derivatives bearing a benzyloxy substituent, for novel MAO-B inhibitors, have been selected as promising scaffolds with potent inhibition.13–20 These MAO-B inhibitors are related to natural compounds, such as coumarins, indoles, chromones, and chromanone analogues (Fig. 1, compounds 1–4).13–16 Interestingly, we find that some smaller molecules also exhibit potent MAO-B inhibitory activity, when the heterocycle of hetero-benzofused derivatives is opened (Fig. 1, compounds 5–8).17–20 Because these MAO-B inhibitors with benzyloxy substituents are substantially similar, we suppose that the simplified substituents on the benzene ring (A) may also have potential MAO-B inhibitory properties, and the different small substituents may affect the MAO-B inhibitory activity. To obtain smaller MAO-B inhibitors with a novel structure, we modify the molecular structure by introducing different substituents (Br, CHO, and OCH₃) at the meta-position of the benzene ring (A) (Fig. 2). To further examine the structure–activity relationships (SAR) against MAO, different substitutions (F, Br and CH₃) were introduced to the benzyloxy ring, and the effects on MAO inhibition were examined.

Fig. 1. Structures of known MAO inhibitors bearing a benzyloxy substituent.

Fig. 2. Design strategy for the novel series of heterocycle-opened derivatives as MAO-B inhibitors.

Results and discussion

Chemistry

The target compounds (9a–g, 10a–g and 11a–g) were efficiently synthesized as shown in Scheme 1. The commercially available 3-bromophenol (9), 3-hydroxybenzaldehyde (10) and 3-methoxyphenol (11) were reacted with the appropriate benzyl bromides in the presence of K₂CO₃ to give the target compounds in good yields (85–97%).

Scheme 1. Syntheses of target compounds 9a–g, 10a–g and 11a–g. Reagents and conditions: (a) K₂CO₃, CH₃CN, reflux, 12 h.

Inhibition of hMAO activity

For compounds 9a–g, 10a–g and 11a–g, the MAO inhibitory activities were tested with iproniazid as a reference.21 The corresponding IC₅₀ values with MAO and the selectivity ratios (IC₅₀ of MAO-A/IC₅₀ of MAO-B) are shown in Table 1. It could be seen that most of the tested compounds (except compounds 11a–g) were selective MAO-B inhibitors with IC₅₀ values in the low micromolar range. Among the synthesized compounds, compound 9e (IC₅₀ = 0.35 μM, SI > 285.7) and compound 10d (IC₅₀ = 0.19 μM, SI = 206.3) were the most potent and selective inhibitors against MAO-B, being about 23.5-fold more and 43.4-fold more active than iproniazid, respectively.

Table 1. hMAO inhibitory activities of the synthesized compounds.

Compounds	R	MAO-A inhibition a (%)	MAO-B IC50 b (μM)	Selectivity index c
9a	—	23	2.73 ± 0.08	>36.6
9b	3-F	37	1.06 ± 0.18	>94.3
9c	4-F	31	0.58 ± 0.12	>172.4
9d	3-Br	27	0.76 ± 0.03	>131.6
9e	4-Br	30	0.35 ± 0.06	>285.7
9f	3-CH3	43	1.98 ± 0.09	>50.5
9g	4-CH3	24	1.53 ± 0.06	>65.4
10a	—	45.6 ± 0.87 μMb	1.96 ± 0.11	23.3
10b	3-F	64.2 ± 0.59 μM	1.21 ± 0.08	53.1
10c	4-F	42.5 ± 1.06 μM	0.93 ± 0.05	45.7
10d	3-Br	28.2 ± 0.85 μM	0.53 ± 0.04	53.2
10e	4-Br	27.9 ± 0.49 μM	0.19 ± 0.07	146.8
10f	3-CH3	51.6 ± 0.53 μM	2.91 ± 0.08	17.7
10g	4-CH3	64.8 ± 0.73 μM	2.53 ± 0.16	25.6
11a	—	25	15.8 ± 0.23	>6.3
11b	3-F	19	12.3 ± 0.79	>8.1
11c	4-F	22	15.6 ± 0.28	>6.4
11d	3-Br	22	19.3 ± 0.51	>5.2
11e	4-Br	25	8.63 ± 0.24	>11.6
11f	3-CH3	23	10.9 ± 0.17	>9.2
11g	4-CH3	26	14.6 ± 0.35	>6.8
Iproniazid	—	6.78 ± 0.35 μM	8.24 ± 0.18	0.82

^aTest concentration is 100 μM.

^bIC₅₀: 50% inhibitory concentration (means ± SEM of three experiments).

^cSelectivity index = IC₅₀ (MAO-A)/IC₅₀ (MAO-B).

Initially, by introducing benzyloxy groups to compounds 9–11, compounds 9a–11a were synthesized. As shown in Table 1, compound 11a, with an OCH₃ substitution at the meta-position of the benzene ring (A), exhibited an IC₅₀ value of 15.8 μM for MAO-B which was less active than the Br-substituted compound 9a (IC₅₀ = 2.73 μM) and the CHO-substituted compound 10a (IC₅₀ = 1.96 μM). From this result, it might be concluded that the electron-withdrawing groups at the meta-position of the benzene ring (A) were more suitable for MAO-B inhibition.

Then, we thought of introducing substituents with varying positions and electronic properties with respect to benzyloxy substitution to study the possible effects on MAO-B inhibition potency. It was noteworthy that those substituted at the para-position (compounds 9c, 9e and 9g) were more potent than those substituted at the corresponding meta-position (compounds 9b, 9d and 9f). Compared to compound 9a, compounds 9b–e bearing electron-withdrawing groups exhibited large enhancement in MAO-B inhibition. However, compounds 9f and 9g with an electron-donating group showed a slight increase in MAO-B inhibition. For example, compound 9e (IC₅₀ = 0.35 μM for MAO-B) possessing the electron-withdrawing substituent Br was about 8-fold more active than compound 9a, while compound 9f (IC₅₀ = 1.98 μM for MAO-B) substituted with CH₃, increased the MAO-B inhibition potency of compound 9a by 1.4-fold. Furthermore, among compounds 9b–g, compounds with para-substitution of the benzyloxy phenyl ring were more potent for MAO-B inhibition than those with meta-substitution.

Moreover, by introducing different substituents to benzyloxy of compound 10a to study the possible effects on MAO inhibition, compounds 10b–g were synthesized. Interestingly, those substituted at the para-position were more potent than those substituted at the corresponding meta-position, which was consistent with compounds 9b–g. Additionally, compared to compound 10a, compounds 10f and 10g with an electron-donating group showed a slight decrease in MAO-B inhibition.

Overall, these results demonstrated that substitution with a wide variety of benzyloxy side chains of small molecules led to structures with potent MAO-B inhibition. Meanwhile, MAO-A inhibition was very weak and no apparent SARs existed.

Reversibility of hMAO-B inhibition

As we know, MAO-B inhibitors could be classified as irreversible and reversible. To examine whether the small molecules bearing a benzyloxy substituent were reversible or irreversible MAO-B inhibitors, the time dependencies of inhibition were evaluated.14 Compounds 9e and 10e were selected as representative inhibitors since they displayed the most potent MAO-B inhibitory activity. For a reversible inhibitor, the MAO-B activity would be almost the same, when the enzyme was preincubated with a reversible inhibitor over different time periods. In contrast, for an irreversible inhibitor, the MAO-B activity would show a time-dependent reduction. Compounds 9e and 10e were preincubated with MAO-B over different time periods (0–60 min) at a concentration of twofold the IC₅₀. As shown in Fig. 3, we could observe that MAO-B activities were almost the same (compound 9e: 44.8% at 0 min, 44.9% at 15 min, 48.6% at 30 min and 49.2% at 60 min; compound 10e: 46.4% at 0 min, 45.8% at 15 min, 49.9% at 30 min and 49.5% at 60 min), and the results demonstrated that compounds 9e and 10e were not time-dependent inhibitors of MAO-B. So these experiments clearly indicated that the small molecules bearing a benzyloxy substituent were reversible MAO-B inhibitors.

Fig. 3. The time-dependent inhibition of hMAO-B by compounds 9e and 10e. The compounds were preincubated for various periods of time (0–60 min) with hMAO-B at concentrations equal to twofold the IC₅₀ values for the inhibition of the enzyme. After dilution to concentrations equal to the IC₅₀, the inhibitory rates were recorded.

Kinetic study of hMAO-B inhibition

Compound 9e was also used to further investigate the mode of MAO-B inhibition. The type of MAO-B inhibition was determined by Michaelis–Menten kinetic experiments.22 The catalytic rates were measured at five different p-tyramine concentrations (50–500 μM), and each plot was constructed at four different concentrations of compound 10e (0, 0.095, 0.19 and 0.38 μM). The overlaid reciprocal Lineweaver–Burk plots (Fig. 4) showed that the plots for different concentrations of compound 9e were linear and intersected at the y-axis. This pattern indicated that compound 9e was a competitive MAO-B inhibitor, and these results further proved that the small molecules bearing a benzyloxy substituent were reversible MAO-B inhibitors.

Fig. 4. Kinetic study on the mechanism of hMAO-B inhibition by compound 10e. Overlaid Lineweaver–Burk reciprocal plots of the MAO-B initial velocity at an increasing substrate concentration (50–500 μM) in the absence of the inhibitor and in the presence of 10e are shown. Lines were derived from a weighted least-squares analysis of the data points.

Molecular modeling studies

In order to explain the difference in MAO-B inhibition, we have carried out a structure-based molecular modeling study using hMAO cocrystals deposited with the PDB. Crystallographic structures of MAO-B (PDB code 2V61) were used to dock the derivatives under study.17 And a molecular docking study was performed using the software package MOE 2008.10.23 According to the inhibition results, compounds 9e, 10e and 11e with different small substituents (Br, CHO, and OCH₃) at the meta-position of the benzene ring (A) were selected as typical ligands. The 3D and 2D pictures of binding are illustrated in Fig. 5. As shown in Fig. 5A and B, compound 9e is located in the well-known binding pocket of MAO-B,24 with the Br-substituted benzene ring (A) interacting with Tyr 398 via aromatic π–π stacking interactions at the bottom of the substrate cavity; the Br-substituted benzyloxy group occupied the entrance cavity via π–π interactions with Tyr 326. From Fig. 5C and D, compound 10e, with the CHO-substituted benzene ring (A), not only had aromatic π–π stacking interactions with Tyr 398, but also had a hydrogen bond interaction with Tyr 188 at the bottom of the substrate cavity; the Br-substituted benzyloxy group occupied the entrance cavity which is a hydrophobic subpocket existing only in the MAO-B isoform and is composed of Leu 171, Ile 316, Tyr 326, Ile 199, Phe 99, Pro 104 and Phe 168. However, no interaction between the ligand 11e with the OCH₃-substituted benzene ring (A) and the MAO-B was observed in Fig. 5E and F. So these results might explain why the MAO-B inhibitory activities of compounds 9e and 10e were more potent than that of compound 11e, and the reason could be ascribed to the different interactions between compounds and the MAO-B. Moreover, the modelling studies between compound 10e and the standard compound 7-((3-chlorobenzyl)oxy)-4-((methylamino)methyl)-coumarin (C. Binda, J. Med. Chem., 2007, 50, 5848–5852) are compared in the ESI.‡

Fig. 5. (A) A 3D docking model of compound 9e with hMAO-B. Atom colors: yellow – carbon atoms of compound 9e, gray – carbon atoms of residues of hMAO-B, dark blue – nitrogen atoms, and red – oxygen atoms. The dashed lines represent the interactions between the protein and the ligand. (B) A 2D schematic diagram of a docking model of compound 9e with hMAO-B. (C) A 3D docking model of compound 10e with hMAO-B. (D) A 2D schematic diagram of a docking model of compound 10e with hMAO-B. (E) A 3D docking model of compound 11e with hMAO-B. (F) A 2D schematic diagram of a docking model of compound 11e with hMAO-B. The figure was prepared using the ligand interactions application in MOE.

Cell toxicity

Based on the screening results above, compounds 9e and 10e as the most potent inhibitors against MAO-B were selected to further examine their potential toxicity effect on SH-SY5Y cells.25 After incubating the cells with compound 9e or 10e for 48 h, the cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. As shown in the Fig. 6, the results revealed that compounds 9e and 10e at 3–50 μM did not have neurotoxicity. This suggested that compounds 9e and 10e might be used to develop promising drug candidates for the therapy of neurodegenerative diseases.

Fig. 6. Effects of compounds on cell viability in SH-SY5Y cells. The cell viability was determined by the MTT assay after 48 h of incubation with various concentrations of 9e and 10e. The results were expressed as a percentage of control cells. Values are reported as the mean ± SD of three independent experiments.

Prediction of BBB penetration of compounds 9a–g, 10a–g and 11a–g

High pharmacological activity and low toxicological effects are not enough for a compound to become a drug candidate. Molecules should meet the limiting terms of Lipinski's rules: a molecular weight (MW) less than 500, the number of hydrogen bond acceptor atoms (HBA) should be less than 10, the number of hydrogen bond donor atoms (HBD) should be less than 5, the calculated logarithm of the octanol–water partition coefficient (clog P) should be less than 5, and a small polar surface area less than 90 Ų. With the aim of developing CNS drugs, the ability of compounds to cross the blood–brain barrier (BBB) is very important.26 log BB is calculated as: log BB = 0.0148 × PSA + 0.152 × clog P + 0.139.27 By calculating their log BB for their potential applications as CNS drugs and with their properties fitting the definition of the restrictive terms of Lipinski's rules, as shown in Table 2, compounds 9a–g, 10a–g and 11a–g satisfied possible brain penetration and drug-like standards.

Table 2. Physical properties of compounds 9a–g, 10a–g and 11a–g.

Compounds	MW a	clog P a	HBA a	HBD a	PAS a	log BB a
9a	261.99	4.832	1	0	9.23	1.010
9b	279.99	4.975	1	0	9.23	1.031
9c	279.99	4.975	1	0	9.23	1.031
9d	339.91	5.695	1	0	9.23	1.141
9e	339.91	5.695	1	0	9.23	1.141
9f	276.02	5.311	1	0	9.23	0.953
9g	276.02	5.311	1	0	9.23	0.953
10a	212.08	3.546	2	0	26.30	1.067
10b	230.07	3.690	2	0	26.30	1.089
10c	230.07	3.690	2	0	26.30	1.089
10d	289.99	4.410	2	0	26.30	1.199
10e	289.99	4.410	2	0	26.30	1.199
10f	226.10	4.046	2	0	26.30	1.143
10g	226.10	4.046	2	0	26.30	1.143
11a	214.10	3.918	2	0	18.46	0.878
11b	232.09	4.061	2	0	18.46	1.029
11c	232.09	4.061	2	0	18.46	1.029
11d	292.01	4.781	2	0	18.46	1.139
11e	292.01	4.781	2	0	18.46	1.139
11f	228.12	4.417	2	0	18.46	1.084
11g	228.12	4.417	2	0	18.46	1.084
Rules	≤450	≤5.0	≤10	≤5	≤90	≥–1.0

^aMW: molecular weight; clog P: calculated logarithm of the octanol–water partition coefficient; HBA: hydrogen-bond acceptor atoms; HBD: hydrogen-bond donor atoms; PSA: polar surface area; log BB = 0.0148 × PAS + 0.152 × clog P + 0.139.

Conclusions

In conclusion, we have synthesized a series of small molecules bearing a benzyloxy substituent and evaluated their MAO inhibitory activity and toxicity in vitro. The results showed that most of the studied compounds were remarkably competitive and reversible inhibitors of MAO-B rather than of MAO-A. The SAR of the synthesized compounds showed that the electron-withdrawing groups at the meta-position of the benzene ring (A) were more suitable for MAO-B inhibition; halogen substituents on the benzyloxy ring further increased MAO-B inhibition. So different electron-withdrawing groups will be introduced at different positions of the benzene ring (A) and the MAO-B inhibition will be tested, which is an on-going further study. Moreover, molecular docking studies of 9e and 10e suggested that the potent MAO-B inhibition might be ascribed to the π–π stacking/cation–π interactions and the larger set of residues interacting with MAO-B. Due to the possible BBB permeability and low neurotoxicity in SH-SY5Y cells in vitro, these compounds could be used to develop promising drug candidates for the therapy of neurodegenerative diseases.

Experimental section

Materials and methods

All chemicals (reagent grade) used were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). The reaction progress was monitored using analytical thin layer chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qing-Dao, China) plates and the spots were detected under UV light (254 nm). ¹H NMR and ¹³C NMR spectra were measured on a BRUKER AVANCE III spectrometer at 25 °C and referenced to TMS. Chemical shifts are reported in ppm (δ) using the residual solvent line as an internal standard. Splitting patterns are designed as s, singlet; d, doublet; t, triplet; and m, multiplet. The purity of all compounds was confirmed to be higher than 95% through analytical HPLC performed with an Agilent 1200 HPLC System, with a photodiode array detector (DAD), 55% (v/v) of CH₃OH gradient, and a flow rate of 1.0 mL min^–1. Mass spectra were obtained on a MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS).

General procedure for the preparation of compounds 9a–g, 10a–g and 11a–g

Compounds 9, 10 or 11 (1.85 mmol) was suspended in acetonitrile (15 mL) containing K₂CO₃ (3.70 mmol). The reaction was treated with an appropriately substituted arylalkyl bromide (2.04 mmol) and heated under reflux for 12 h. The reaction progress was monitored using silica gel TLC with hexanes/EtOAc as the mobile phase. Upon completion, the acetonitrile was evaporated in vacuo and the mixture was then poured into water, which was extracted with 3 × 200 mL of EtOAc, washed with brine, dried over anhydrous Na₂SO₄ and purified by chromatography (hexanes/EtOAc) on silica gel.

1-(Benzyloxy)-3-bromobenzene (9a)

Yield 89%; light yellow oil; ESI/MS m/z: 264.1 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 7.45–7.38 (m, 4H), 7.37–7.31 (m, 1H), 7.18–7.07 (m, 3H), 6.95–6.87 (m, J = 8.0, 2.4, 1.2 Hz, 1H), 5.05 (s, 2H). ¹³C-NMR (100 MHz, CDCl₃): δ 159.57, 136.42, 130.59, 128.68, 128.18, 127.51, 124.10, 122.84, 118.22, 113.86, 70.24.

1-Bromo-3-((3-fluorobenzyl)oxy)benzene (9b)

Yield 92%; light yellow oil; ESI/MS m/z: 282.1 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.36–7.25 (m, 1H), 7.20–7.09 (m, 5H), 7.03–6.95 (m, 1H), 6.89–6.81 (m, 1H), 5.04 (s, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ 162.93 (d, ¹J_CF = 287.43 Hz), 159.24, 139.01 (d, ³J_CF = 8.27 Hz), 130.64, 130.22 (d, ³J_CF = 8.48 Hz), 124.35, 122.88, 122.71, 118.21, 115.01 (d, ²J_CF = 21.68 Hz), 114.23 (d, ²J_CF = 22.25 Hz), 113.80, 69.37.

1-Bromo-3-((4-fluorobenzyl)oxy)benzene (9c)

Yield 93%; light yellow oil; ESI/MS m/z: 282.1 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.35 (m, 2H), 7.18–7.03 (m, 5H), 6.89–6.79 (m, 1H), 5.00 (s, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ 162.62 (d, ¹J_CF = 245.43 Hz), 159.37, 132.18, 132.15, 130.62, 129.38 (d, ³J_CF = 8.36 Hz), 124.24, 122.87, 118.19, 115.59, 115.59 (d, ²J_CF = 21.59 Hz), 113.83, 69.56.

1-Bromo-3-((3-bromobenzyl)oxy)benzene (9d)

Yield 89%; light yellow oil; ESI/MS m/z: 343.0 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.58 (s, 1H), 7.47 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.29–7.23 (m, 1H), 7.18–7.08 (m, 3H), 6.89–6.80 (m, 1H), 5.01 (s, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ 159.22, 138.73, 131.23, 130.66, 130.35, 130.22, 125.84, 124.39, 122.90, 122.76, 118.21, 113.76, 69.28.

1-Bromo-3-((4-bromobenzyl)oxy)benzene (9e)

Yield 90%; light yellow oil; ESI/MS m/z: 343.1 [M + H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 7.59 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.28–7.20 (m, 2H), 7.14 (d, J = 7.9 Hz, 1H), 7.02–6.95 (m, 1H), 5.11 (s, 2H). ¹³C NMR (101 MHz, DMSO-d6) δ 159.58, 136.55, 131.86, 131.86, 131.69, 130.30, 130.30, 124.23, 122.57, 121.55, 118.16, 114.80, 69.16.

1-Bromo-3-((3-methylbenzyl)oxy)benzene (9f)

Yield 87%; light yellow oil; ESI/MS m/z: 278.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.29 (t, J = 7.5 Hz, 1H), 7.25–7.20 (m, 2H), 7.18–7.08 (m, 4H), 6.91–6.86 (m, 1H), 5.00 (d, J = 6.0 Hz, 2H), 2.38 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 159.63, 138.39, 136.31, 130.57, 128.95, 128.57, 128.27, 124.63, 124.04, 122.83, 118.21, 113.83, 70.31, 21.43.

1-Bromo-3-((4-methylbenzyl)oxy)benzene (9g)

Yield 86%; light yellow oil; ESI/MS m/z: 278.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.31 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 7.8 Hz, 2H), 7.17–7.05 (m, 3H), 6.90–6.83 (m, 1H), 5.00 (s, 2H), 2.37 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 159.64, 138.00, 133.36, 130.55, 129.35, 129.35, 127.66, 127.66, 123.99, 122.81, 118.21, 113.87, 70.18, 21.22.

3-(Benzyloxy)benzaldehyde (10a)

Yield 88%; light yellow oil; ESI/MS m/z: 213.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 9.98 (s, 1H), 7.50–7.32 (m, 8H), 7.26–6.17 (m, 1H), 5.13 (s, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ 192.07, 159.35, 137.86, 136.33, 130.13, 128.70, 128.70, 128.22, 127.55, 127.55, 123.69, 122.21, 113.32, 70.26.

3-((3-Fluorobenzyl)oxy)benzaldehyde (10b)

Yield 93%; light yellow oil; ESI/MS m/z: 231.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 9.98 (s, 1H), 7.52–7.44 (m, 3H), 7.36–7.24 (m, 1H), 7.27–7.14 (m, 3H), 7.03 (td, J = 8.3, 2.4 Hz, 1H), 5.12 (s, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ 191.97, 163.02 (d, ¹J_CF = 245.48 Hz), 159.05, 138.93 (d, ³J_CF = 7.83 Hz), 137.89, 130.30, 130.22, 123.99, 122.79, 122.17, 115.06 (d, ²J_CF = 21.32 Hz), 114.27 (d, ²J_CF = 22.14 Hz), 113.14, 69.38.

3-((4-Fluorobenzyl)oxy)benzaldehyde (10c)

Yield 90%; light yellow oil; ESI/MS m/z: 231.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 9.98 (s, 1H), 7.50–7.45 (m, 3H), 7.45–7.39 (m, 2H), 7.27–7.22 (m, 1H), 7.12–7.06 (m, 2H), 5.08 (s, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ 192.02, 162.64 (d, ¹J_CF = 245.58 Hz), 159.17, 137.87, 132.08, 130.18, 129.43, 129.43 (d, ³J_CF = 8.27 Hz), 123.92, 122.22, 115.62, 115.62 (d, ²J_CF = 21.84 Hz), 113.11, 69.57.

3-((3-Bromobenzyl)oxy)benzaldehyde (10d)

Yield 90%; light yellow oil; ESI/MS m/z: 292.1 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 9.95 (s, 1H), 7.59 (s, 1H), 7.49–7.45 (m, 2H), 7.45–7.42 (m, 2H), 7.34 (d, J = 7.7 Hz, 1H), 7.25–7.20 (m, 2H), 5.06 (s, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ 191.95, 159.03, 138.65, 137.90, 131.28, 130.39, 130.24, 130.24, 125.90, 124.01, 122.79, 122.14, 113.15, 69.29.

3-((4-Bromobenzyl)oxy)benzaldehyde (10e)

Yield 91%; light yellow oil; ESI/MS m/z: 292.1 [M + H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 7.61 (s, 1H), 7.50–7.47 (m, 2H), 7.45–7.41 (m, 2H), 7.35 (d, J = 7.7 Hz, 1H), 7.26–7.21 (m, 2H), 5.21 (s, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ 193.35, 159.09, 139.97, 138.15, 131.25, 131.17, 130.94, 130.72, 130.27, 127.09, 123.43, 122.15, 114.38, 68.95.

3-((3-Methylbenzyl)oxy)benzaldehyde (10f)

Yield 89%; light yellow oil; ESI/MS m/z: 227.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 9.98 (s, 1H), 7.51–7.45 (m, 3H), 7.30–7.22 (m, 4H), 7.16 (d, J = 7.3 Hz, 1H), 5.09 (s, 2H), 2.39 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 192.10, 159.41, 138.43, 137.86, 136.23, 130.12, 129.00, 128.60, 128.32, 124.68, 123.62, 122.18, 113.36, 70.33, 21.43.

3-((4-Methylbenzyl)oxy)benzaldehyde (10g)

Yield 91%; light yellow oil; ESI/MS m/z: 227.1 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 9.97 (s, 1H), 7.51–7.43 (m, 3H), 7.34 (d, J = 8.0 Hz, 2H), 7.27–7.19 (m, 3H), 5.09 (s, 2H), 2.37 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 192.10, 159.41, 138.05, 137.84, 133.29, 130.10, 130.10, 129.38, 129.38, 127.71, 123.57, 122.23, 113.37, 70.21, 21.22.

1-(Benzyloxy)-3-methoxybenzene (11a)

Yield 91%; light yellow oil; ESI/MS m/z: 215.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.60 (s, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 7.7 Hz, 1H), 7.29–7.16 (m, 3H), 6.56–6.43 (m, 3H), 5.02 (s, 2H), 3.80 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 160.91, 159.75, 139.36, 131.03, 130.38, 130.15, 130.00, 125.88, 122.70, 106.90, 106.87, 101.44, 69.13, 55.31.

1-Fluoro-3-((3-methoxyphenoxy)methyl)benzene (11b)

Yield 93%; light yellow oil; ESI/MS m/z: 233.2 [M + H]⁺; 1H NMR (400 MHz, CDCl₃) δ 7.60 (s, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 7.7 Hz, 1H), 7.29–7.16 (m, 2H), 6.56–6.41 (m, 3H), 5.02 (s, 2H), 3.80 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 163.02 (d, ¹J_CF = 245.35 Hz), 160.91, 159.78, 139.65 (d, ³J_CF = 8.27 Hz), 130.13 (d, ³J_CF = 8.24 Hz), 129.99, 122.76, 114.80 (d, ²J_CF = 21.33 Hz), 114.26 (d, ²J_CF = 22.26 Hz), 106.93, 106.81, 101.44, 69.21, 55.30.

1-((4-Fluorobenzyl)oxy)-3-methoxybenzene (11c)

Yield 91%; light yellow oil; ESI/MS m/z: 233.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.45–7.36 (m, 2H), 7.23–7.16 (m, 1H), 7.12–7.02 (m, 2H), 6.60–6.50 (m, 3H), 5.01 (s, 2H), 3.79 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 162.54 (d, ¹J_CF = 245.19 Hz), 160.89, 159.89, 132.73, 129.96, 129.38, 129.38 (d, ³J_CF = 8.95 Hz), 115.50, 115.50 (d, ³J_CF = 22.11 Hz), 106.94, 106.71, 101.43, 69.37, 55.29.

1-Bromo-3-((3-methoxyphenoxy)methyl)benzene (11d)

Yield 90%; light yellow oil; ESI/MS m/z: 294.1 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.45 (m, 2H), 7.42–7.37 (m, 2H), 7.20 (t, J = 8.1 Hz, 1H), 6.62–6.51 (m, 3H), 5.06 (s, 2H), 3.80 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 160.88, 160.10, 137.01, 129.93, 128.62, 128.62, 128.00, 127.55, 127.55, 106.99, 106.64, 101.43, 70.08, 55.30.

1-((4-Bromobenzyl)oxy)-3-methoxybenzene (11e)

Yield 94%; light yellow oil; ESI/MS m/z: 294.1 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.58 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 7.18 (t, J = 8.1 Hz, 1H), 6.60–6.54 (m, 2H), 6.53 (dd, J = 8.2, 2.1 Hz, 1H), 5.07 (s, 2H), 3.72 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6) δ 160.97, 159.83, 137.07, 131.81, 131.81, 130.46, 130.22, 130.22, 121.35, 107.46, 107.04, 101.60, 68.84, 55.56.

1-Methoxy-3-((3-methylbenzyl)oxy)benzene (11f)

Yield 91%; light yellow oil; ESI/MS m/z: 229.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.36–7.13 (m, 5H), 6.59–6.47 (m, 3H), 5.04 (s, 2H), 3.82 (s, 3H), 2.41 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 160.87, 160.16, 138.30, 136.89, 129.90, 128.77, 128.51, 128.31, 124.67, 106.95, 106.60, 101.40, 70.14, 55.29, 21.43.

1-Methoxy-3-((4-methylbenzyl)oxy)benzene (11g)

Yield 91%; light yellow oil; ESI/MS m/z: 229.3 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.36 (d, J = 8.0 Hz, 2H), 7.22 (t, J = 8.2 Hz, 3H), 6.67–6.52 (m, 3H), 5.04 (s, 2H), 3.82 (s, 3H), 2.40 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 160.85, 160.16, 137.76, 133.94, 129.88, 129.28, 129.28, 129.28, 129.28, 106.98, 106.55, 101.40, 69.99, 55.28, 21.21.

Abbreviations

5-HT

5-Hydroxy-tryptamine

BBB

Blood–brain barrier

CNS

Central nervous system

DA

Dopamine

FAD

Flavin adenine dinucleotide

MAO

Monoamine oxidase

MTT

Methyl thiazolyl tetrazolium

NE

Norepinephrine

SI

Selectivity index

Conflicts of interest

The authors declare no conflict of interest.